全豆乳液体发酵制备纳豆激酶工艺优化
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摘要
纳豆激酶具多效溶栓作用,是一种理想的预防和治疗血栓的天然药物。液体发酵可提高纳豆激酶产量。以纳豆枯草芽胞杆菌为出发菌株,豆乳为发酵基质,纳豆激酶活力为核心评价指标,出渣率和终点pH为辅助评价指标,通过单因素和多因素统计学分析研究发酵时间、装液量、接菌量和初始pH对纳豆激酶活力的影响,优化发酵条件,以期提升纳豆激酶的表达量。最终确定最优发酵工艺为:摇瓶豆乳的发酵时间30 h,装液量5%,接菌量1.75%,初始pH 5.5。在该发酵条件下,纳豆激酶的活力为429 869.34 IU/mL,是优化前的5.41倍。可见,正交试验成功优化了纳豆枯草芽孢杆菌的液体发酵工艺,显著提升了纳豆激酶的表达量,为功能食品开发奠定了理论基础。
Nattokinase is an ideal natural drug for the prevention and treatment of thrombosis. Liquid fermentation can improve the yield of nattokinase. Taking Bacillus subtilis natto as the start culture, soybean milk as the fermentation medium, nattokinase activity as function evaluation index, the slag rate and finial pH as auxiliary indexes, the effects of fermentation time, liquid loading, bacteria loading and initial pH on nattokinase activity were studied by single factor and orthogonal analysis. The optimal fermentation conditions were determined as follows: The fermentation time was 30 h, the liquid volume in flask was 5%, the amount of bacteria was 1.75%, and the initial pH was 5.5. The nattokinase activity was 429 869.34 IU/mL under this condition, which was 5.41 times of that before optimization. It can be seen that the orthogonal test successfully optimized the liquid fermentation process of Bacillus subtilis natto and significantly increased the expression of nattokinase, which laied a theoretical foundation for the development of functional food.
Nattokinase is an ideal natural drug for the prevention and treatment of thrombosis. Liquid fermentation can improve the yield of nattokinase. Taking Bacillus subtilis natto as the start culture, soybean milk as the fermentation medium, nattokinase activity as function evaluation index, the slag rate and finial pH as auxiliary indexes, the effects of fermentation time, liquid loading, bacteria loading and initial pH on nattokinase activity were studied by single factor and orthogonal analysis. The optimal fermentation conditions were determined as follows: The fermentation time was 30 h, the liquid volume in flask was 5%, the amount of bacteria was 1.75%, and the initial pH was 5.5. The nattokinase activity was 429 869.34 IU/mL under this condition, which was 5.41 times of that before optimization. It can be seen that the orthogonal test successfully optimized the liquid fermentation process of Bacillus subtilis natto and significantly increased the expression of nattokinase, which laied a theoretical foundation for the development of functional food.
引文
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[3] 李淑英,辛凤姣,王凤忠.纳豆激酶,一种潜在的用于预防与治疗心血管疾病的功能食品添加剂[J].生物产业技术,2017(6):100-106.
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[8] 明飞平,赵培静,廖春芳,等.纳豆芽孢杆菌液体发酵条件的优化研究[J].现代食品科技,2009,25(06):625-628.
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[11] 卓晓沁,赵慧莹,何国庆.鹰嘴豆纳豆液态发酵高产蛋白酶的培养基及发酵条件优化[J].食品工业科技,2018,(7):115-121.
[12] Nie G, Liu N, Zhang E, et al.. Preparation of a novel mixed milk with nattokinase produced by Bacillus subtilis (natto)[J]. J. Food Proc. Preserv., 2017, 41(6):e13284.
[13] 赵仲麟, 李淑英, 聂莹, 等. 纳豆激酶生产菌株分离筛选与热稳定性分析[J]. 生物技术进展, 2013,3(4):277-280.
[14] Astrup T, Mullertz S. The fibrin plate method for estimating fibrinolytic activity[J]. Arch. Biochem. Biophys., 1952, 40(2):346-351.
[15] 奚晓琦,王加启,卜登攀, 等.纳豆芽孢杆菌的分离鉴定及纳豆激酶高产菌株的筛选[J].东北农业大学学报,2009(11):69-75.
[16] 吴燕,李英波,梁向峰, 等.纳豆激酶液态发酵生产的研究进展[J].食品与发酵工业,2017(10):256-262.
[17] 王丽娜.纳豆芽孢杆菌液体发酵条件的优化[J].中国调味品,2014(9):28-30.
[18] Ravichandra P, Subhakar C, Annapurna J. Alkaline protease production by submerged fermentation in stirred tank reactor using Bacilluslicheniformis NCIM-2042: Effect of aeration and agitation regimes [J]. Biochem. Eng. J.,2007, 34(2):185-192.
[19] 朱向辉. 纳豆液体发酵条件优化研究[D]. 长春:吉林大学, 硕士学位论文,2006.
[20] Rasagnya P S,Vangalapa T M. Studies on optimization of process parameters for nattokinase production by Bacillus subtilis NCIM 2724 and purification by liquid-liquid extraction [J].Int. J. Innov. Res. Sci., Engin. Technol.,2013,2:4516-4521.
[2] Tai M W, Sweet B V. Nattokinase for prevention of throm bosis[J]. Am. J. Health Syst. Pharm., 2006, 63(12):1121-1123.
[3] 李淑英,辛凤姣,王凤忠.纳豆激酶,一种潜在的用于预防与治疗心血管疾病的功能食品添加剂[J].生物产业技术,2017(6):100-106.
[4] Lampe B J, English J C. Toxicological assessment of nattokinase derived from Bacillus subtilis var. natto[J]. Food Chem. Toxicol., 2016, 88:87-99.
[5] 王爱萍,林燕飞,王赞,等.纳豆激酶的研究综述[J].科技视界,2018(12):110-111.
[6] Tyagi M G. Nattokinase enzyme; an evaluation of its cellular and potential therapeutic actions[J]. Eur. J. Pharm. Med. Res., 2016,3(1): 411-414.
[7] 史旭东,孙丹,肖兴龙,等.纳豆菌株Ⅰ液体发酵生产纳豆激酶发酵条件的优化[J].食品工业,2012,33(9):101-102.
[8] 明飞平,赵培静,廖春芳,等.纳豆芽孢杆菌液体发酵条件的优化研究[J].现代食品科技,2009,25(06):625-628.
[9] Kitamura Y, Jianfeng S, Takahashi S, et al.. Growth characteristics of Bacillus subtilis (natto) in milk.[J]. Biken J., 2010, 63(3):418-422.
[10] 吴昱含,郭大鹏,李帅鹏, 等.液态发酵豆粕制备纳豆激酶方法的优化[J].生物资源,2017,(4):308-313.
[11] 卓晓沁,赵慧莹,何国庆.鹰嘴豆纳豆液态发酵高产蛋白酶的培养基及发酵条件优化[J].食品工业科技,2018,(7):115-121.
[12] Nie G, Liu N, Zhang E, et al.. Preparation of a novel mixed milk with nattokinase produced by Bacillus subtilis (natto)[J]. J. Food Proc. Preserv., 2017, 41(6):e13284.
[13] 赵仲麟, 李淑英, 聂莹, 等. 纳豆激酶生产菌株分离筛选与热稳定性分析[J]. 生物技术进展, 2013,3(4):277-280.
[14] Astrup T, Mullertz S. The fibrin plate method for estimating fibrinolytic activity[J]. Arch. Biochem. Biophys., 1952, 40(2):346-351.
[15] 奚晓琦,王加启,卜登攀, 等.纳豆芽孢杆菌的分离鉴定及纳豆激酶高产菌株的筛选[J].东北农业大学学报,2009(11):69-75.
[16] 吴燕,李英波,梁向峰, 等.纳豆激酶液态发酵生产的研究进展[J].食品与发酵工业,2017(10):256-262.
[17] 王丽娜.纳豆芽孢杆菌液体发酵条件的优化[J].中国调味品,2014(9):28-30.
[18] Ravichandra P, Subhakar C, Annapurna J. Alkaline protease production by submerged fermentation in stirred tank reactor using Bacilluslicheniformis NCIM-2042: Effect of aeration and agitation regimes [J]. Biochem. Eng. J.,2007, 34(2):185-192.
[19] 朱向辉. 纳豆液体发酵条件优化研究[D]. 长春:吉林大学, 硕士学位论文,2006.
[20] Rasagnya P S,Vangalapa T M. Studies on optimization of process parameters for nattokinase production by Bacillus subtilis NCIM 2724 and purification by liquid-liquid extraction [J].Int. J. Innov. Res. Sci., Engin. Technol.,2013,2:4516-4521.